Brazilian Journal of Microbiology (2005) 36:196-200 ISSN 1517-8382

HISTOPATHOLOGY OF HÜBNER (; NOCTUIDAE) TREATED WITH NUCLEOPOLYHEDROVIRUS AND BACILLUS THURINGIENSIS SEROVAR KURSTAKI

Neiva Knaak*; Lidia Mariana Fiuza*

Laboratório de Microbiologia, Universidade do Vale do Rio dos Sinos, São Leopoldo, RS, Brasil

Submitted: December 17, 2002; Returned to authors for corrections: June 03, 2004; Approved: May 27, 2005

ABSTRACT

The Anticarsia gemmatalis is responsible for the use of chemical insecticides in the soybean culture, causing a significant increase in the costs of farming and a great unbalance in the ecosystem. The use of microbial agents, like Bacillus thuringiensis serovar kurstaki (Btk) and Anticarsia gemmatalis nucleopolyhedrovirus (AgNPV), they are an alternative to chemical control of the pest . In the interaction analysis of the entomopathogenic bacteria and virus it is considered important the in vitro action mode of these microbiology control agents. Therefore, the present study aims the histopathological analysis of the A. gemmatalis larvae digestive system after the interaction in vivo of the entomopathogenic Btk and AgNPV, represented the Dipel and Baculovirus anticarsia formulations, respectively. The evaluations were realized in larvae of 2nd instar, in which the mortality was evaluated daily, and a histopathology was done with collected larvae in time of 1, 3, 6, 12 and 24 hours after the treatments application. The results of the in vivo assays reveal that the treatment using the association of AgNPV-Btk (98.68% of mortality) was more efficient than using AgNPV isolatedly (81.28% of mortality), but the Btk when used isolatedly had a mortality of 100%. The treatments showed significant (P<0.05) differences between AgNPV and Btk, AgNPV and AgNPV/Btk. The histopathological analysis of the AgNPV and Btk in A. gemmatalis larvae suggests that the Dipel and Baculovirus anticarsia products were more efficient when they were used simultaneously, because the action of AgNPV was intensified when used in association with Btk, causing changes in the larvae midgut after 6 hours of treatments. When the entomopathogens were used isolating the gut cells alterations were observed only 12 hours after the treatments.

Key words: virus, bacteria, biological control, Lepidoptera

INTRODUCTION aerobic or facultative anaerobic entomopathogenic bacterium found in soil, on plant surfaces, and in grain storage dust. During The velvetbean caterpillar Anticarsia gemmatalis Hübner sporulation B. thuringiensis produce crystalline protein (Lepidoptera: Noctuidae) is an important pest of the inclusions (i.e. crystal). When ingested by insects, this crystal is soybean fields in Brazil. The larva cause serious damage by dissolved in the midgut, discharging proteins denominated delta- reducing the leaf area greatly and, consequently, reducing endotoxins or Cry proteins. Those proteins after hydrolysis have photosynthesis and productivity (18). specific toxin activity for insects and other invertebrate, not Among the alternatives to control this pest, the use of Bacillus causing harmful effects in other organism (25). In the midgut, the thuringiensis has gained attention due to its efficiency and low proteases activate these proteins that interact with the epithelial impact on natural enemies (3). B. thuringiensis is a Gram-positive, membrane, causing the insect death (6). In the field, as in the

*Corresponding Author. Mailing address: Laboratório de Microbiologia, Universidade do Vale do Rio dos Sinos, Av. Unisinos, 950. 93001-970, São Leopoldo, RS, Brasil. Fax: (+5551) 591-1100, E-mail: [email protected]; [email protected]

196 Histopathology of A. gemmatalis

soybean farming’s that agent of biological insects control is used mL; for AgNPV and 2.5.107 cells/mL; for Btk) dilutions were in formulations as the Dipel product (Abbott Laboratories of made and the exact number of bacterial cells and viral Brasil Ltda). polyhedrons were determined in Neubauer chamber. After the In the microbial control context, the Anticarsia gemmatalis treatments application, 20 insects were individually nucleopolyhedrovirus (AgNPV) stands out as the most conditioned, in which four repetitions were accomplished by important virus used in the insects biological control (5). In treatment. The mortality was daily evaluated until the 7th day natural conditions, the curse is infected commonly when after the treatments application and the corrected mortality ingesting the contaminated food. After the ingestion, the bodies was calculated according to Abbott (1). Data were subjected of polyhedral inclusion, finding alkaline conditions in the either to one-way analysis of variance (ANOVA), TUKEY 5% mesenterum are dissolved, liberating the virions. The virus and factorial analysis of variance (FANOVA) depending upon begins multiplying itself in the nucleus cells of the insect, the experimental design. dispersing itself for the whole insect body, provoking death, usually from 6 to 8 days after the ingestion (12,24). Insects’ tissues To understand the mode action of entomopathogenic The A. gemmatalis larva were prepared according to the bacteria and virus, it is important to knowledge of the insect inclusion paraffin techniques. During the bioassay, 20 larvae histopatology. The insect digestive system’s is one main were used for each treatment and 20 to witness, in which they physiochemical barriers against the pathogenic agents. In this were collected in periods of 1, 3, 6, 12 and 24 hours, after the way, the morphology and physiochemical aspects can support treatments application. After the fixation, in Bouin Hollande to understand of the insect defense mechanism’s (16). Sublime (BHS) for 24 hours (7), the tissues were submitted to Like this, the present study aimed the histopathological dehydration in ethanol solutions in growing order of graduation analysis of the A. gemmatalis larvae digestive system, after the (70, 96 and 100%), following by fast baths of xylol and interaction in vivo of the entomopathogenic Bacillus impregnation in paraffin. The longitudinal histological cuts were thuringiensis kurstaki and Anticarsia gemmatalis accomplished to 5µm thickness, in the histology laboratory nucleopolyhedrovirus, representing the formulations Dipel (UNISINOS). To remove the paraffin, the slide containing the (Abbott) and Baculovirus anticarsia (Embrapa-CNPSo), tissue passed by xylol and ethanol baths in decreasing order of respectively. graduation. The staining, of the tissues of A. gemmatalis was made with Heidenhain’s Blue. The glasses were mounted with MATERIALS AND METHODS Etellan. The longitudinal sections of the gut tissues of the A. gemmatalis larvae were observed under direct optical Insects microscopy were amplified 400 times. The Anticarsia gemmatalis were obtained from soybean fields in the Rio Grande do Sul state and maintained in RESULTS AND DISCUSSION laboratory in the Ciências da Saúde of the Universidade do Vale do Rio dos Sinos (UNISINOS). The insects were maintained Pathogenicity in vivo in the laboratory at 70% relative humidity, photoperiod of 12 The evaluations of the pathogenicity were considering hours and 25ºC. Larvae were reared with artificial diet prepared the Anticarsia gemmatalis corrected mortality medium, in according to Greene et al. (14). which the treatment with Anticarsia gemmatalis nucleopolyhedrovirus (AgNPV) was verified a mortality of Bioassay 81.28%, in the association Btk/AgNPV, the mortality was The bioassays were accomplished in the same laboratory 98.68% and only Bacillus thuringiensis serovar kurstaki (Btk) and on controlled conditions (25ºC, 70% of Relative humidity was equivalent to 100%. The results, in vivo, reveal that and 12 hours of photoperiod), using larvae of 2nd instar of A. regarding association treatment AgNPV/Btk was more efficient gemmatalis, conditioned in acrylic mini-plates (35mm of than only AgNPV, however only Btk caused a similar mortality. diameter), in which was previously applied Greene’s diet (14). The in vivo toxicity obtained in this study using 2.5.107 cells/ In assays were used in the treatments Anticarsia gemmatalis mL of the Btk HD1 against the target species was similar to nucleopolyhedrovirus (AgNPV) and Bacillus thuringiensis those obtained by Bobrowski et al. (8 and 9) using Btk HD73 serovar kurstaki (Btk), strain HD1. The products were obtained and Btk UNI872 strains. from A. gemmatalis Baculovirus (EMBRAPA/CNPSo) and Dipel Considering the lethal time, through the daily analysis of (Abbott Laboratories of Brazil Ltda.), respectively. Were applied the bioassay, it was observed that the treatment with Btk on the in the artificial diet the treatments Btk; AgNPV; Btk + AgNPV 3rd day after the treatment (DAT) caused 96.05% of mortality, and witness (100 µL of NaCl 0.85% solution). Considering the keeping like this until 7th DAT. As for the treatment with AgNPV, treatments, to reach the wanted concentration (3.107 particles/ in 3rd DAT there was a mortality of 25% that raised itself to

197 N. Knaak and L.M. Fiuza

61.05% in 7th DAT. In the association Btk/AgNPV, the mortality observed the desruption of microvilli and a part of the cells was of 45% in 2nd DAT, with a peak of 85% in 3rd DAT, causing was already disorganized and lysed. Bacillus thuringiensis 90.26% of mortality in 7th DAT (Fig. 1). The treatments showed toxins thus appear to bind specifically on the apical membrane significant (P<0.05) differences in AgNPV and Btk, AgNPV and of the Lymantria dispar (Lepidoptera, Lymantriidae) larvae AgNPV/Btk. epithelial cells (21). According to this study, Raussel et al. These results can indicate that the natural resistance factor (22) were observed the histopathology changes in the midgut is very important at the moment of applying the biopesticide of the 3rd instar of Lymantria monacha larvae (Lepidoptera, against A. gemmatalis larvae. Showing that the control’s Lymantriidae) when treated with B. thuringiensis toxins, integrate method, using Btk HD1 (Dipel) and AgNPV causing vacuolization of the cytoplasm and increase of the (Baculovirus), is a good alternative to reduce the problems cellular volume. Similar effects were observed by Bobrowski concerning the resistance to the population of target pest. et al. (10): disruption of microvilli and vacuolization of Janmaat et al. (15) mentioned that the continued use of the Bt microbial pesticide was directly correlated with the concentration’s increase of Bt applied by treatment. According to Abot et al. (2), A. gemmatalis has a high potential of developing resistance to the Anticarsia gemmatalis AgNPV in Brazil, where the virus occurs naturally and is extensively employed as a microbial pesticide in the soybean fields. In the United States, the potential for resistance interfering with a microbial insecticide based on AgNPV is lower. Resistant insects also had longer life spans, a lower rate of larval survival in rearing and lower pupa weights than susceptible insects (13).

Histopathology in vitro The observations in optical microscopy analysis, of the midgut of A. gemmatalis larvae, when compared with the witness (Fig. 2A), show changes in the structure of the midgut, after 24 hours of the treatment with Btk (Fig. 2C), where was

Figure 2. Longitudinal sections of the midgut Anticarsia gemmatalis larvae treated with: (A) Control, not treated; (B) 24 hours after the treatment with Baculovirus anticarsia and Bacillus thuringiensis serovar kurstaki; (C) 24 hours after treatment with Figure 1. Lethal time of Anticarsia gemmatalis evaluated daily, Bacillus thuringiensis kurstaki; (E) Epithelium; (L) Lumen; (MV) after the application of the nucleopolyhedrovirus (AgNPV) and Microvilli; ( ) Effects caused by the treatments. The slides were Bacillus thuringiensis serovar kurstaki (Btk) treatments. amplified 400x.

198 Histopathology of A. gemmatalis

cytoplasm began 6 hours after B. thuringiensis ingestion by The in vitro interaction between AgNPV and Btk showed a A. gemmatalis larvae. Despite the lower toxicity of Cry1C, difference significant (P <0.05) when compared whit the synthesized by B. thuringiensis serovar kurstaki HD1 strain treatments isolated. The pathogenicity analysis of AgNPV and used in the Dipel biopesticide, which lacks Cry1C, it increased Btk in A. gemmatalis larvae suggests that the Dipel and toxicity to Spodoptera exigua (Lepidoptera, Noctuidae). These Baculovirus anticarsia products were more efficient when used indicate the half of Cry1C was synthesized effectively in this simultaneously, because the action of AgVPN was intensified strain, a finding that may increase these strains commercial when used in association with Btk, causing changes in the utility (19). According to Aranda et al. (4), 15 minutes after the larvae midgut after 6 hours of treatments. Only 12 hours after application of Cry 1C and Cry 1D in S. frugiperda, it was the treatments, and when the entomopathogens were used already possible to observe changes in their cells, however isolating, were observed the gut cells alterations. the vacuolization of the cytoplasm, the degradation of the membrane and the destruction of the cells are observed 1 hour RESUMO after the treatments. An increase of the cellular volume, vacuolization of the Histopatologia de Anticarsia gemmatalis Hübner cytoplasm, ejection of a great number of cells in the midgut (Lepidoptera; Noctuidae) tratadas com Virus de and disruption on the microvilli were observed in the Poliedrose Nuclear e Bacillus thuringiensis application of AgNPV (Fig. 2B), after 24 hours of the treatment. sorovar kurstaki Similar results were described by several researches. According to Matos et al. (17) although the virus were not found in the A Anticarsia gemmatalis é responsável pelo uso de nuclei of columnar cells until late on infection, it is believed inseticidas químicos na cultura da soja, ocasionando um that these cells are the primary sites of infection and replication. significativo aumento nos custos das lavouras e um grande This fact can be explained by the continuous regeneration of desequilíbrio no ecossistema. O uso de agentes microbianos, the midgut epithelium. Besides, the infection may be occurring como Bacillus thuringiensis sorovar kurstaki (Btk) e Vírus de in isolated cells. Pombo et al. (20) observed that 12 hours post Poliedrose Nuclear de Anticarsia gemmatalis (VPNAg), é uma infection of A. gemmatalis with AgMNPV, the cells became alternativa para o controle químico de insetos-praga. Na análise round and exhibited a decrease in the number of cytoplasmic da interação de bactérias e vírus entomopatogênicos, considera- projections. The authors described that by 24 hours it was se importante o modo de ação in vitro desses agentes de controle possible to detect a virogenic stroma inside the cell nucleus microbiano. Assim, o presente trabalho objetiva a análise and after 48 hours, polyhedral inclusion bodies were observed. histopatológica do sistema digestivo das lagartas de A. According to this present study, the Castro et al. (11) gemmatalis, após a interação dos entomopatógenos Btk e evaluated infection of the permissive A. gemmatalis cell line VPNAg, representados nas formulações Dipel e Baculovirus with AgNPV showed pronounced cytopathic effect by 48 anticarsia, respectivamente. As avaliações foram realizadas com hours. These cells were rounded and developed nuclear lagartas de 2º ínstar, onde a mortalidade foi avaliada diariamente, hypertrophy. Same data was observed by Sciocco-Cap et al. e a histopatologia foi realizada com lagartas coletadas nos (23), showing in the histological analysis of the 4th instar of períodos de 1, 3, 6, 12 e 24 horas após a aplicação dos tratamentos. Epinotia aporema larva (Lepidoptera, Tortricidae) infected Os resultados dos ensaios in vivo, revelam que o tratamento with Baculovirus that were presented the same phases of referente à associação VPNAg/Btk (98.68% de mortalidade) foi cellular disruption. mais eficiente que VPNAg (81.28% de mortalidade) isoladamente, In the Btk/AgNPV association, it was observed that after 6 porém o Btk isoladamente causou 100% de mortalidade. Os hours of the application, it had begin disintegrate of the tratamentos mostraram diferenças significativas (P <0,05) entre peritrophic membrane and brush border membrane. The cellular AgNPV e Btk, AgNPV e AgNPV/Btk. As análises de lyse was greater and the lumen presented amount of cells after patogenicidade do VPNAg e Btk em lagartas de A. gemmatalis 12 hours of the treatments and this one was intensified after 24 sugerem que os produtos Dipel e Baculovirus anticarsia foram hours. Those observations demonstrated that, in the individual mais eficientes, quando utilizados simultaneamente, pois a ação treatments with Btk and AgNPV, until 6 hours after application do VPNAg foi intensificada quando utilizada em associação com of the treatments, there were no alterations in the intestinal Btk, provocando alterações no intestino médio das lagartas a cells of the A. gemmatalis larva. However, Btk and AgNPV in partir de 6 horas após os tratamentos. Quando os association, after 6 hours already presented intense entomopatógenos foram utilizados isoladamente, as alterações vacuolization of the cytoplasm, causing cellular disorganization. das células intestinais foram observadas apenas 12 horas após After 12 and 24 hours all treatments revealed the alterations (i.e. a aplicação dos tratamentos. increase of the nucleus) in the midgut cellular structure of the velvetbean caterpillar. Palavras-chave: vírus, bactéria, controle biológico, Lepidoptera

199 N. Knaak and L.M. Fiuza

REFERENCES 14. Greene, G.L.; Leppla, N.C.; Dickerson, W.A. Velvetbean caterpillar a rearing procedure and artificial medium. J. Econ. Entomol., 69, 1. Abbott, W.S. A method of computing the effectiveness insecticides. 487-488, 1976. J. Econ. Entomol., 18, 265-267, 1925. 15. Janmaat, A.F.; Myers,J. Bacillus thuringiensis resistance and 2. Abot, A.R.; Moscardi, F.; Fuxa, J.R.; Sosa-Gómez, D.R.; Richter, associated fitness costs in Trichoplusia ni populations. VIII A.R. Development of resistance by Anticarsia gemmatalis from International colloquium on Invertebrate pathology and microbial Brazil and the United States to a Nuclear Polyhedrosis Virus Under control/XXXV Annual meeting of the SIP/VI International Laboratory Selection Pressure. Biol. Control., 7, 126-130, 1996. Conference on Bacillus thuringiensis, Foz do Iguassu, 2002, p. 67. 3. Alves, S.B. Controle microbiano de insetos. Piracicaba: Ed. FEALQ, 16. Levy, S.M.; Gregório, E.A.; Falleiros, A.M.F.; Arrebola, N.R. Estudo 1998, p. 1163. Ultraestrutural do tubo digestivo de Anticarsia gemmatalis. VII 4. Aranda, E.; Sanchez, J.; Peferoen, M.; Güereca, L.; Bravo, A. Simpósio de controle Biológico, Poços de Caldas, 2001, p. 237. Interactions of Bacillus thuringiensis crystal proteins with the midgut 17. Matos, T.G.T.; Giugliano, L.G.; Bergmann, M.R.; Báo, S.N. Structural epithelial cells of Spodoptera frugiperda (Lepidoptera:Noctuidae). and ultrastructural studies of Anticarsia gemmatalis midgut cells J. Invertebr. Pathol., 68, 203-212, 1996. infected with the baculovirus A. gemmatalis nucleopolyhedrovirus. 5. Azevedo, J.L. Controle microbiano de insetos-praga e seu Int. J. Insect. Morphol. Embryol., 28, 195-201, 1999. melhoramento genético. In: Azevedo, L.J.; Melo, I.S. (ed). Controle 18. Morales, L.; Moscardi, F.; Kastelic, J.G.; Sosa-Gomez, D.R.; Paro, biológico. Embrapa, São Paulo, 1998, p. 69-96. F.R.; Soldorio, I.L. Suscetibilidade de Anticarsia gemmatalis Hübner 6. Barreto, M.R.; Boregas, K.G.B.; Paiva, E.; Valicente, F.H. Banco de e Chrysodeixis includens (Walker) (Lepidoptera: Noctuidae), a da Embrapa milho e sorgo: Bacillus thuringiensis. VII Simpósio de Bacillus thuringiensis (Berliner). Annais da Sociedade Entomológica controle biológico, Poços de Caldas, 2001, p. 75. do Brasil, 24, 1044-1050, 1995. 7. Brandtzaeg, P. Tissue preparation methods for immunocytochemistry. 19. Park, H.W.; Bideshi, D.K.; Federici, B.A. Molecular genetic In: Bullock, G.; Petruz, P. (eds) Techniques in immunocytochemistry. manipulation of truncated Cry1C protein synthesis in Bacillus Academic Press, London, 1982, p. 49-51. thuringiensis to improve and yield. Appl. Environ. Microbiol., 66, 8. Bobrowski, V.L.; Pasquali, G.; Bodanese-Zanettini, M.H.; Fiuza, L.M. 4449-4455, 2000. Detection of CRY1 genes in Bacillus thuringiensis isolates from 20. Pombo, V.; Velloso, L.M.; Bergmann, M.R.; Báo, S.N. Structural and south of Brazil and activity against Anticarsia gemmatalis ultrastructural changes during the infection of UFL-AG-286 cells (Lepidoptera:Noctuidae). Braz. J. Microbiol., 32, 105-109, 2001. with the Baculovirus AgMNPV. J. Invertebr. Pathol., 72. 239-245, 9. Bobrowski, V.L.; Pasquali, G.; Bodanese-Zanettini, M.H.; Fiuza, L.M. 1998. Characterization of two Bacillus thuringiensis isolates from south 21. Peyronnet, O.; Vachon, V.; Brousseau, R.; Baines, D.; Schwartz, J.L.; Brazil and their toxicity against Anticarsia gemmatalis Laprade, R. Effect of Bacillus thuringiensis toxins the membrane (Lepidoptera:Noctuidae). Biol. Control., 25, 129-135, 2002. potential of lepidopteran insect midgut cells. Appl. Environ. 10. Bobrowski, V.L.; Scheneumann, R.; Pasquali, G.; Bodanese-Zanettini, Microbiol., 63, 1679-1684, 1997. M.H.; Fiuza, L.M. Characterization of Bacillus thuringiensis aizawai 22. Rausell, C.; Decker, N.; García-Robles, I.; Esriche, B.; Van Kerkhove, UNI498 and histopathology analysis of the toxic effect on midgut E.; Real, M.D.; Martinez-Ramirez, A.C. Effect of Bacillus of the Anticarsia gemmatalis. VIII International Colloquium on thuringiensis toxins on the midgut of the nun Lymantria Invertebrate Pathology and Microbial Control/XXXV. Annual monacha. J. Invertebr. Pathol., 75, 288-291, 2000. meeting of the SIP/VI International Conference on Bacillus 23. Sciocco-Cap, A.; Parola, A.D.; Goldberg, A.V.; Chiringhelli, P.D.; thuringiensis, Foz do Iguassu, 2002, p.49. Romanowski, V. Characterization of a Granulovirus Isolated from 11. Castro, M.E.B.; Souza, M.L.; Araujo, S.; Bilimoria, S.L. Replication Epinotia aporema Wals. (Lepidoptera: Tortricidae) Larvae. Appl. of Anticarsia gemmatalis Polyhedrosis Virus in Four Lepidopteron Environ. Microbiol., 8, 3702-3706, 2001. Cell Lines. J. Invertebr. Pathol., 69, 40-45, 1997. 24. Silva, M.T.B. Associação de Baculovírus anticarsia com subdosagem 12. Cruz, I. Utilização do Baculovírus no controle da lagarta-do-cartucho de inseticidas no controle de Anticarsia gemmatalis (Hübner,1818). do milho, Spodoptera frugiperda. In: Azevedo, L.J.; Melo, I.S. (ed.). Ciência Rural, 3, 353-358, 1995. Controle Biológico. Embrapa, São Paulo, 2000, p. 201-204. 25. Silva-Werneck, J.O.; Dantas, A.S.; Oliveira, J.A.; Araújo, T.M.R.; 13. Fuxa, J.R.; Richter, A.R. Repeated reversion of resistance to Praça, S.F.; Monnerat, R.G. Caracterização de isolados de Bacillus Nucleopolyhedrovirus by Anticarsia gemmatalis. J. Invertebr. thuringiensis efetivos contra Lepidópteros-praga. VII Simpósio de Pathol., 71, 159-164, 1998. Controle Biológico, Poços de Caldas, 2001, p. 104.

200